Eliminating cancer from a patient's body is challenging because, although cancerous cells proliferate in an uncontrolled manner, the cells do not necessarily appear to be “foreign” to the body and are therefore difficult to target. Existing cancer treatments tend to be insufficiently targeted to the cancer cells and are destructive to a patient's healthy tissue. Such treatments typically include X-rays, chemotherapy, proton therapy, and surgery. Treatments that incite the body's immune system to exhibit a positive immune response against these cancer cells would be preferred.
Although cancer cells express cancer-associated antigens, they are often able to evade an immune response because of their ability to hide the cancer antigens from the immune system and/or because the exposed antigens are normal, non-mutated differentiation molecules or proteins that the human immune system normally recognizes or tolerates. Cancer stem cells also have been reported that are resistant to current therapies of chemotherapy and radiation. (See, e.g., Targeted therapy for cancer stem cells: the patched pathway and ABC transporters. Oncogene, (2007) 26(9):1357-60.; Radiation resistance and stem-like cells in brain tumors. Cancer Cell (2006); 10(6):454-6.; WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA (2007) 104(2):618-23. Epub Jan. 3, 2007)
To effectively use immunotherapy to treat a cancer, a patient must have, or be provided with, a sufficient number of cancer-reactive lymphocytes that can both reach the cancer site and have effector mechanisms to destroy the cancer cells.
Some therapies under investigation are aimed at heightening the immune response in general, and include for example administration of chemical messengers such as cytokines (e.g. IL-2 and/or IL-12), lymphocytes specific for telomerase, bacterial extracts or drugs that boost the immune system. In an attempt to make the immune response more specific for the tumor cells, some treatments administer autologous tumor cells either combined with cytokines—e.g. GM-CSF, gamma interferon or IL-2, individually or in combination—or transfected with the genes that encode these cytokines. Some success has been observed in cell-transfer therapies where autologous lymphocytes are sensitized to cancer cells ex vivo and then infused back into the patient. A similar approach utilizes tumor cell lines instead of autologous tumor cells.
Adjuvants are commonly used with cancer vaccine immunotherapy. One approach uses dendritic cells (DCs) that are highly potent antigen-presenting cells to provoke a positive anti-cancer immune response in patients. Dendritic cells express MHC class I and MHC class II molecules, co-stimulatory molecules and adhesion molecules that provide signals for the stimulation of naive T cells, CD4+ T-helper cells, CD8+ cytotoxic T lymphocytes (CTLs), natural killer (NK) and thymic derived NK cells (NKT) cells. DCs have the capacity to take up various types of molecules. Consequently, DCs can be loaded with tumor-associated antigens (TAAs) in various forms and administered as vaccines.
One DC-based approach uses DC-cancer cell hybrids generated by fusion of cancer cells with dendritic cells to combine sustained cancer antigen expression with the antigen-presenting and immune stimulatory capabilities of the DC. In animal models, immunization with DC-cancer cell hybrids can provide some form of anti-cancer protection or eradicate established disease. Hybrids of autologous DCs comprised of cancer cell lines or primary human cancer cells (including breast carcinoma cells) have been shown to induce CTL responses against autologous cancer cell types in vitro. Clinical studies of the treatment of renal cell carcinoma and glioma have demonstrated that vaccination with DC-cancer cell hybrids can safely induce anti-cancer immune responses in patients.
One hypothesis to explain how tumors grow and metastasize is the cancer stem cell hypothesis, which states that there is a small, distinct subset of cells within each tumor that is capable of indefinite self-renewal and of developing into the more adult tumor cell(s), which are relatively limited in replication capacity. It has been hypothesized that these cancer stem cells (CSC) might be more resistant to chemotherapeutic agents, radiation or other toxic conditions, and thus, persist after clinical therapies and later grow into secondary tumors, metastases or be responsible for relapse. It has been suggested that CSCs can arise either from the tissue stem cells or from a more differentiated tissue progenitor cell(s). While supporting data for this is strong for hematopoietic stem and progenitor cells and hematopoietic tumors, less is known about solid tumors and their respective CSCs.
Solid tumors are thought to arise in organs that contain stem cell populations. The tumors in these tissues consist of heterogeneous populations of cancer cells that differ markedly in their ability to proliferate and form new tumors; this difference in tumor-forming ability has been reported for example with breast cancer cells and with central nervous system tumors. While the majority of the cancer cells have a limited ability to divide, recent literature suggests that a population of cancer cells, termed cancer stem cells, has the exclusive ability to extensively self-renew and form new tumors. Growing evidence suggests that pathways that regulate the self-renewal of normal stem cells are deregulated or altered in cancer stem cells, resulting in the continuous expansion of self-renewing cancer cells and tumor formation.
It has been suggested that cancer patient prognosis is associated with stem cell phenotype/biology. (See e.g., Molecular profiling identifies prognostic subgroups of pediatric glioblastoma and shows increased YB-1 expression in tumors. J Clin Oncol. (2007) 25(10):1196-208; Cancer stem cells are central to metastasis, which accounts for 90% of the lethality of cancer. Cell Res. (2007) 17:3-14.) It has also been observed that patients with autoimmune reactions to self-stem cells demonstrate decreased cancer progression. (See, e.g. “immunity to cancer stem cells may help protect people with a precancerous condition from developing the full-blown disease” J Exp Med (2007) 204(4):831-40.)
Tissue stem cells exist in specific niches or microenvirouments that are critical for maintaining them in the appropriate developmental and metabolic state. These microenvironments are not completely understood, but their disruption by genetically knocking out an important factor can result in the disregulation of stem cell homeostasis both during development and in the adult. In trying to understand the microenvironments that support tissue stem/progenitor cells, many researchers have taken the approach of deriving serum free culture conditions where the medium, substrate and physical environment produce an optimized environment for maintaining specific fetal and neonatal tissue stem/progenitor cells (SPC) in a defined state in which the SPC can replicate, but not differentiate (see for example U.S. Pat. Nos. 6,436,704 and 6,416,999). The optimized media and culture conditions that are distinct for different types of SPC can be seen as recreating the stem cell niche that these cells occupy in vivo. These media and optimized conditions are specifically tailored to the SPCs in that they specifically select out the SPCs and cannot support the survival and/or growth of any other cell types in a tissue. Consequently, non-SPC cells will not survive and replicate under the SPC-preferred conditions and are lost during culture and passage, leaving only a pure SPC population, even when the SPC represents only a very small percentage of the starting culture. The conditions must also remove any signals for further differentiation of the SPC to allow its maintenance in culture over an extended period of time.
One hypothesis about how tumors originate is that tumors arise from the tissue SPC by a series of mutational events. Data exists to suggest that some tumor cells will “home in” to specific SPC niches when they move about the body during the process of metastasis. Media and optimized culture conditions derived to support the survival and growth of SPC might also be able to preferentially support the growth and survival of CSCs, thus selecting for this type of cell when the dispersed tumors are put into culture. Such media and optimized culture conditions may allow for the establishment of pure CSC cultures that would be capable of long-term or extensive growth and maintenance of the characteristics of the rare CSC phenotype.
The tumor stem cell has been hypothesized, but there does not yet exist a reliable way of identifying these cells, nor does consensus exist on all their characteristics. Some researchers have proposed that cancer stem cells can be identified based on marker expression (see e.g., Al-Hajj et al. (2003) Proc Natl Acad Sci USA 100:3983-3988; O'Brien et al. (2007) Nature 445:106-110; and Clarke et al. (2006) Cell 124:1111-5). CD133 has been proposed to be a marker found in cancer stem cells in brain tumors and in human prostatic epithelial stem cells. CD44 expression accompanied by no or low CD24 expression was hypothesized to be expressed by some solid cancer stem cells (e.g., breast cancer). CD34 is a marker present on the surface of blood vessels and immature blood cells that has also been associated with hematopoietic stem cells.
The establishment of pure CSC cultures would be a great advantage in studying and the understanding of the regulation of tumorgenesis and metastasis and in the discovery and development of CSC-directed therapies for cancer. Accordingly, there exists a need for methods to identify, isolate, culture and characterize cancer stem cells.
The invention described herein overcomes many of the unmet needs and shortcomings mentioned above and provides for methods of isolation, maintenance and growth of human cancer stem cells. The invention also describes a constellation of characteristics of cancer stem cells, and specifically the characteristics of a novel population of cells from colon, prostate, lung, pancreas, breast, mantle cell lymphoma, and Merkel's tumors with the biological characteristics of CSCs.
The present invention also provides a simple, effective and efficient method for treating cancer, preventing cancer, delaying the onset of cancer or delaying the progression of cancer via administration of the CSC-based vaccines and treatments described herein.